The present invention relates to plasma collection and, more particularly, to a device and method for separating and discharging plasma. The field of plasma collection plays an important role in analytical methods for determining a concentration of blood components. In many cases such blood tests cannot be carried out with whole blood since this contains corpuscular components (blood cells) which could result in an interference of the assay procedure. Hence, in order to carry out many analytical methods it is necessary to firstly isolate plasma from whole blood, which plasma should be as free as possible from cellular material.
A conventional method for isolating plasma for blood tests is the centrifugation procedure in which cellular components of the blood are separated on the basis of centrifugal forces. This method is laborious and is especially unsuitable when only small amounts of plasma are required for an analysis. However, particularly modem miniaturized tests use quantities of plasma that are only in the range of a few microliters. This applies especially to so-called carrier-bound tests in which an analytical system that is as small and compatible as possible is present, for example, in the form of a test strip. In this case all reagents and other agents required to carry out the test are integrated into the test strip. In order to determine an analyte the sample liquid is contacted with such an analytical element. The reagent contained in the test element reacts within a short period with the analyte to be determined such that a physically detectable change occurs on the analytical element. Such a change can, for example, be a colour change or a change in a measurable electrical variable. The change is measured and calculated with the aid of an evaluation instrument in order to output an analytical result.
An example of an analytical system which determines an analyte from plasma by means of such a test element is the HDL test (high density lipoproteins). The determination of the HDL concentration in blood is, among other things, important for the risk assessment of coronary heart disease and is thus in recent times used to diagnose one of the most common modem diseases.
The severity of a coronary heart disease can be assessed on the basis of several known parameters such as total cholesterol, in the blood, plasma or serum. Since the concentration of total cholesterol is of only limited use for individual risk assessment, the low density lipoproteins (LDL) and the high density lipoproteins (HDL) are quantified separately from one another in modern analytical methods. When assessing such analytical methods it must be taken into account that there is a positive correlation between LDL cholesterol and a coronary heart disease but a negative correlation between HDL cholesterol and the disease. Clinical studies have proven that, as a first approximation, the determination of HDL and total cholesterol is sufficient for a risk assessment. This is the preferred method in current diagnostic practice.
HDL cholesterol is, for example, determined by means of an analytical element such as those known in the prior art (e.g., HDL test elements from Roche Diagnostics GmbH). Since the HDL cholesterol is determined separately, the other lipoprotein classes that are present have to be separated from the remaining blood components to allow the determination of HDL cholesterol in plasma. Such a test, for example, requires a plasma volume of about 40 μl in order that the concentration can be determined independently of the applied plasma volume. Only pure plasma in which there are substantially no blood components can be used to determine HDL cholesterol. A complexing agent which is also integrated into the test element is additionally used to determine the HDL concentration. Plasma is then applied to the zone of the test element in which the complexing agent is present. The complexing agent EDTA is, for example, used in the prior art to analyze HDL cholesterol.
However, an analyte in pure plasma can also be determined by means of a test element that requires no complexing agent to determine an analyte. Such test elements are, for example, used to determine enzymes and are described in the prior art in the document DE 3130749 among others. Test elements which determine an analyte in pure plasma but do not contain a complexing agent are often designed such that the test element itself separates plasma. For this purpose such test elements contain a separation layer in addition to a reagent layer. In order to measure an analyte whole blood is firstly applied to the separation layer. Blood components are separated from the plasma within the separation layer and the plasma is passed on to the reagent layer. In this manner an analyte can be determined in pure plasma although blood has been applied to the test element. However, such a plasma separation layer integrated into the test element cannot be used when using complexing agents. If a complexing agent is used to determine an analyte, it turns out that the complexing agent in the test element prevents the separation of plasma from blood. Hence, the test element can no longer separate plasma if the test element contains a complexing agent.
Since, on the one hand, a complexing agent is needed to determine HDL cholesterol by one of the test elements described above and, on the other hand, it is necessary to separate plasma from blood, it follows that the plasma has to be already separated from blood on a μl scale before applying blood to the test element.
In this connection the determination of HDL cholesterol is only one important example of an analyte determination which requires small amounts of pure plasma for analysis. Other fields of application for the use of released plasma are in the field of clinical analysis. Since test strips are currently preferably used in diagnostic practice as analytical systems, there is an increasing need for simple methods for obtaining small amounts of plasma in order to achieve an overall simplification and more rapid analytical procedure.
In this respect several methods have been described in the prior art which are intended to simplify the isolation of small amounts of plasma. The aim is to obtain plasma from likewise small volumes of blood to spare the patients a laborious blood withdrawal which would, for example, be required for a centrifugation method.
Filtration methods in which different filter media and in particular membrane and glass filters are used have been discussed for many years and in some cases have been used successfully. Earlier examples of filtration technology are described in U.S. Pat. Nos. 3,791,933 and 4,477,575. A recent example comprising a complicated combination of membrane and glass filters is described in U.S. Pat. No. 5,922,210. Small amounts of plasma are obtained by microfiltration with the aid of a microcomponent. The blood cells are separated by a so-called barrier channel which is too small to allow blood cells to flow through the barrier channel. However, manufacture of a device with such a channel requires special manufacturing processes which are complicated. The said plasma collection methods have the additional disadvantage that there is a high risk of the fine pores being clogged up by mechanical plugging or by accumulation of cellular material on the walls of the pores. This would reduce the filter capacity. However, an enlargement of the filter capacity would require more space for the filter medium. This would in turn have an unfavorable effect in relation to the applied sample volume and volume of plasma obtained.
A filtering process for collecting plasma is described in U.S. Pat. No. 4,477,575 which comprises a glass fiber layer which improves the relation between sample volume and the volume of plasma obtained. In this case the volume of plasma to be separated is preferably less than 30% of the suction volume of the glass fiber layer. The filtering process takes place after blood has been applied to the glass fiber layer and is driven solely by gravity and hence the plasma isolation is correspondingly time consuming.
In order to accelerate plasma collection other methods for collecting plasma have been described in the prior art. In the patent application EP 747105 a glass fiber onto which blood is applied is firstly stored in a vessel. Pressure is exerted by a plunger on the glass fiber and the blood contained therein to accelerate the filtration process. The blood is thus pressed through the glass fiber resulting in a separation of plasma from other blood components. The plasma is discharged via an outlet. However, a disadvantage of the described device is that large amounts of blood sample are required due to the filtering process. In addition, pressing out the glass fibers results in a destruction of the corpuscular blood components and hence it is not possible to obtain pure plasma.
A vessel for plasma collection, which is described in the patent application EP 0 785 012, is based on a similar principle. In this case pressure is also exerted on a filter material to which blood has previously been applied to allow plasma separation to occur. As already described above, the pressing out process destroys blood cells and hence pure plasma is not obtained. Once plasma has been contaminated by the destruction of blood cells, it is unsuitable for use in numerous analytical tests.